Cell-fusion techniques have been devised for several eucaryotic cell systems to facilitate formation of hybrid cells. Genetic exchange mediated by cell fusion had not been demonstrated with procaryotic microorganisms until Shaeffer et al. and Fodor and Alfoldi devised techniques involving fusion of protoplasts and regeneration of cells of the procaryotic genus Bacillus, [P. Schaeffer et al., Proc. Nat. Acad. Sci. 73, 2151-2155 (1976) and K. Fodor and L. Alfoldi, Proc. Nat. Acad. Sci. 73, 2147-2150 (1976)]. Still more recently, it was discovered that protoplastfusion-induced genetic exchange, including genetic recombination, is possible within the economically important genus Streptomyces [Baltz and Godfrey, copending patent application titled METHOD OF FACILITATING GENETIC EXCHANGE IN STREPTOMYCES BY PROTOPLAST FUSION Ser. No. 812,097, filed this even date].
A crucial step in the process of protoplast-fusion-induced genetic exchange or genetic recombination is recovery of variable cells from the fused protoplasts. In the case of Bacillus, the fused protoplasts were reverted to viable cells under conditions that had been well worked out previously for single cells. In the case of the filamentous Streptomyces, however, very little was known about the process of forming protoplasts capable of reverting to viable cells. M. Okanishi, et al. [J. Gen. Microbiol. 80, 389-400 (1974)] reported that S. griseus and S. Venezuelae protoplasts could be formed from cells taken from a mid-exponential growth phase by treatment with lysozyme and lytic enzyme No. 2 in a hypertonic medium and that these protoplasts could regenerate viable cells efficiently. They also reported that cells from stationary phase did not form protoplasts well at all. In contrast to Okanishi's report, I have discovered that Streptomyces cells from the mid-exponential growth phase revert very poorly; but that cells from the transition growth phase, which follows the classical exponential phase but precedes the stationary growth phase, regenerate very efficiently. My discovery relates, therefore, to the optimum conditions for preparing Streptomyces protoplasts which are capable of efficiently resynthesizing cell walls and regenerating viable cells. My technique increases the probability of detecting specific genetic exchange between different Streptomyces strains by protoplast fusion. Genetic exchange is an important tool for increasing variability within species which produce economically and therapeutically important metabolites, such as antibiotics. Industrial applications of this tool include constructing strains which produce high levels of specific metabolites such as antibiotics, antitumor agents, enzymes and other microbial products having useful properties, and constructing hybrid species which produce novel metabolites with useful properties.